SRM/MRM assay for the insulin receptor protein

Abstract

Specific peptides, and derived ionization characteristics of the peptides, from the Insulin Receptor protein (IR), and its isoforms IR-A and IR-B, that are particularly advantageous for quantifying the IR protein, IR-A isoform and/or IR-B isoform, directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the IR protein, and IR-A and/or IR-B isoforms, is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an IR peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

The invention claimed is: 
     
       1. A method for measuring the level of the Insulin Receptor (IR) protein, isoforms IR-A and IR-B in a biological sample of human formalin-fixed tissue, comprising detecting and quantifying the amount of an IR-A fragment peptide and an IR-B fragment peptide protease digest prepared from said biological sample using mass spectrometry; and calculating the level of IR-A isoform and IR-B isoform protein in said sample; wherein said IR-A fragment peptide is the peptide of SEQ ID NO:3 and said IR-B fragment peptide is the peptide of SEQ ID NO:5, and
 wherein said amount is an absolute amount. 
 
     
     
       2. The method of  claim 1 , further comprising the step of fractionating said protein digest prior to detecting and quantifying the amount of said IR fragment peptides. 
     
     
       3. The method of  claim 2 , wherein said fractionating step is selected from the group consisting of liquid chromatography, nano-reversed phase liquid chromatography, high performance liquid chromatography, and reverse phase high performance liquid chromatography. 
     
     
       4. The method of  claim 1 , wherein said protease digest comprises a trypsin digest. 
     
     
       5. The method of  claim 1 , wherein said mass spectrometry comprises triple quadrupole mass spectrometry. 
     
     
       6. The method of  claim 5 , wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring (mSRM), or any combination thereof. 
     
     
       7. The method of  claim 1 , wherein the tissue is paraffin embedded tissue. 
     
     
       8. The method of  claim 1 , wherein the tissue is obtained from a tumor. 
     
     
       9. The method of  claim 1 , wherein quantifying said IR fragment peptides comprises determining the amount of the each of said IR fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the IR fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. 
     
     
       10. The method of  claim 9 , wherein the internal standard peptide is an isotopically labeled peptide. 
     
     
       11. The method of  claim 10 , wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from  18 O,  17 O,  34 S,  15 N,  13 C,  2 H or combinations thereof. 
     
     
       12. The method of  claim 1 , wherein detecting and quantifying the amount of said IR fragment peptides in the protein digest indicates the presence of IR-A and/or IR-B, and an association with cancer in a patient or subject. 
     
     
       13. The method of  claim 12 , wherein quantifying the amount of said IR fragment peptides, or the amount of said IR-A isoform and/or IR-B isoform, is used to diagnose the stage/grade/status of the cancer. 
     
     
       14. The method of  claim 13 , further comprising detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer. 
     
     
       15. The method of  claim 1 , further comprising selecting for a patient or subject from which said biological sample was obtained a treatment based on the presence, absence, or amount of one or more of said IR fragment peptides or the amount of the IR-A isoform and/or IR-B isoform. 
     
     
       16. The method of  claim 15 , further comprising administering to a patient or subject from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon the amount of said IR fragment peptides or the amount of the IR-A isoform and/or IR-B isoform. 
     
     
       17. The method of  claim 16 , wherein the treatment or the therapeutic agent is directed to cancer cells expressing IR protein, IR-A isoform and/or IR-B isoform. 
     
     
       18. A method of determining the resistance of a cancer to an antagonist of IGF-1R, comprising determining the presence or level of IR-A, or the ratio of IR-A to IR-B in a sample of formalin-fixed cancer tissue; wherein the presence of IR-A or an increased ratio of IR-A to IR-B relative to control tissue is indicative of a resistance of said cancer to said antagonist of IFG-IR, wherein the presence or level of IR-A is measured by detecting and quantifying by mass spectrometry the amount of the peptide of SEQ ID NO:3 in a protease digest prepared from said sample, and calculating the level of IR-A, and wherein the presence or level of IR-B is measured by detecting and quantifying by mass spectrometry the amount of the peptide of SEQ ID NO:5 in a protease digest prepared from said sample. 
     
     
       19. The method of  claim 18 , wherein said antagonist of IGF-1R comprises a protein or peptide that binds to the IGF-1R. 
     
     
       20. The method of  claim 19 , wherein said protein or peptide is a: human antibody; humanized antibody; chimeric antibody; monoclonal antibody; monospecific antibody; recombinant antibody; antigen-binding antibody fragment; single chain antibody; diabody; triabody; tetrabody; Fab fragment; F(ab′)2 fragment; domain antibody; IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody. 
     
     
       21. The method of  claim 20 , wherein said protein or peptide is an antibody selected from R1507, OSI-906 and figitumumab.